Werner Syndrome Protein Participates in a Complex with RAD51, RAD54

Home , RAD51, RAD54B, Werner syndrome helicase

Erratum Werner syndrome protein participates in a complex with RAD51, RAD54, RAD54B and ATR in response to ICL-induced replication arrest Marit Otterlei, Per Bruheim, Byungchan Ahn, Wendy Bussen, Parimal Karmakar, Kathy Baynton and Vilhelm A. Bohr

Journal of Cell Science 119, 5215 (2006) doi:10.1242/jcs.03359 There was an error in the first e-press version of the article published in J. Cell Sci. 119, 5137-5146.

The first e-press version of this article gave the page range as 5114-5123, whereas it should have been 5137-5146.

We apologise for this mistake. Research Article 5137 Werner syndrome protein participates in a complex with RAD51, RAD54, RAD54B and ATR in response to ICL-induced replication arrest

Marit Otterlei1,2,*, Per Bruheim1,3,§, Byungchan Ahn1,4,§, Wendy Bussen5, Parimal Karmakar1,6, Kathy Baynton7 and Vilhelm A. Bohr1 1Laboratory of Molecular Gerontology, National Institute on Aging, NIH, 5600 Nathan Shock Dr., Baltimore, MD 21224, USA 2Department of Cancer Research and Molecular Medicine, Laboratory Centre, Faculty of Medicine, Norwegian University of Science and Technology, Erling Skjalgsons gt. 1, N-7006 Trondheim, Norway 3Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway 4Department of Life Sciences, University of Ulsan, Ulsan 680-749, Korea 5Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, 333 Cedar St, SHM-C130, New Haven, CT 06515, USA 6Department of Life Science and Biotechnology, Jadavpur University, Kolkata-700 032, WB, India 7Research Center, Ste. Justine’s Hospital, 3175 Cote Ste. Catherine, Montreal, Quebec H3T 1C5, Canada *Author for correspondence (e-mail: [email protected]) §These authors contributed equally to this work

Accepted 5 October 2006 Journal of Cell Science 119, 5137-5146 Published by The Company of Biologists 2006 doi:10.1242/jcs.03291

Summary Werner syndrome (WS) is a rare genetic disorder resonance energy transfer (FRET) and immune- characterized by genomic instability caused by defects in precipitation experiments to demonstrate that WRN the WRN gene encoding a member of the human RecQ participates in a multiprotein complex including RAD51, helicase family. RecQ helicases are involved in several DNA RAD54, RAD54B and ATR in cells where replication has metabolic pathways including homologous recombination been arrested by ICL. We verify the WRN-RAD51 and (HR) processes during repair of stalled replication forks. WRN-RAD54B direct interaction in vitro. Our data Following introduction of interstrand DNA crosslinks support a role for WRN also in the recombination step of (ICL), WRN relocated from nucleoli to arrested replication ICL repair. forks in the nucleoplasm where it interacted with the HR protein RAD52. In this study, we use ﬂuorescence Key words: WRN, ICL, RAD51, RAD54B, ATR, DNA repair Journal of Cell Science

Introduction The RecQ helicases display 3Ј to 5Ј unwinding directionality Individuals with Werner syndrome (WS) are afflicted with a on DNA on a variety of structures including recombination genomic instability disorder derived from defects in the WRN intermediates such as Holliday junctions (HJ) (Constantinou et gene encoding a member of the RecQ DNA helicase family al., 2000). The WRN protein is unique among human RecQ (Martin, 1978; Yu et al., 1996). RecQ helicases are highly helicases in that it also contains a 3Ј to 5Ј exonuclease activity conserved from bacteria to human; however, bacteria possess a (Huang et al., 1998). How the WRN helicase and exonuclease single RecQ enzyme, whereas in humans, five RecQ members function to resolve DNA structures in vivo is currently unclear; have been identified so far (Hickson, 2003). Deficiencies in three however, cooperativity between the two activities has been of the human RecQ helicases, WRN (Martin et al., 1970), BLM suggested to occur in some situations (Opresko et al., 2003; and RECQL4, are responsible for Werner syndrome (WS), Opresko et al., 2001; Opresko et al., 2004). Bloom syndrome (BS), and Rothmund-Thomson syndrome WRN interacts physically and functionally with a number of (RTS), respectively. All three disorders are characterized by proteins involved in different DNA metabolic pathways decreased genomic stability leading to increased cancer including replication protein A (RPA) (Brosh et al., 1999), incidence (Hickson, 2003). WS is further classified as a proliferating cell nuclear antigen (PCNA) (Lebel et al., 1999), segmental progeroid syndrome since patients display the early polymerase ␦ (Kamath-Loeb et al., 2000), flap endonuclease 1 onset of many age-associated pathologies, such as (FEN-1) (Brosh et al., 2001), RAD52 (Baynton et al., 2003), arteriosclerosis, cataracts, diabetes mellitus type II, greying the MRN complex through binding to NBS1 (Cheng et al., and loss of the hair, wrinkling of the skin, osteoporosis and 2004), the Ku heterodimer (Cooper et al., 2000; Hsu et al., increased predisposition to cancer development, primarily of 2000), and TRF2 (Opresko et al., 2002). Thus, WRN may be mesenchymal origin (Hickson, 2003; Opresko et al., 2003). Cells a multi-tasking enzyme that functions in several genome from WS patients display marked chromosomal rearrangements maintenance pathways, possibly by co-ordinating their and deletions (Fukuchi et al., 1989; Grigorova et al., 2000; Salk, progression. 1982) and a shortened life span in culture (Martin et al., 1970). One process in which WRN function has been strongly The molecular basis of the premature senescence and the implicated is the recovery of arrested replication forks genomic instability in WS cultured cells is not yet clear. (Baynton et al., 2003; Sakamoto et al., 2001). Replication fork 5138 Journal of Cell Science 119 (24)

arrest and collapse may lead to double-strand breaks (DSB) and WRN was recently found to be required for ICL repair in that are repaired in mammalian cells by either non homologous an in vitro assay and thus suggested to be a candidate helicase end joining (NHEJ) or homologous recombination (HR) (for involved in the early steps in processing ICL (Zhang et al., reviews, see Lieber et al., 2003; West, 2003). The essentially 2005). In the early pre-synapsis step of HR, a single-stranded error-free HR repair pathway is currently viewed as the major DNA (ssDNA) tail is converted to a nucleoprotein ﬁlament mechanism for re-establishing stalled replication forks during consisting of RPA, RAD51, RAD52, RAD54 and possibly S-phase in human somatic cells. The current model for the RAD54B (Alexeev et al., 2003; Tan et al., 2003; Tanaka et al., repair and restart of an arrested replication fork involves a 2000). The formation of WRN and RAD52 complexes at structure called a ‘half chicken foot’ or a ‘chicken foot’ arrested replication forks induced by ICL, together with the resembling a Holliday junction that can be resolved by observed stimulation of RAD52 strand annealing activity by recombination (reviewed in Helleday, 2003). Studies in WRN in vitro, suggests that WRN may function both during Escherichia coli indicate that the ‘chicken foot’ can be early steps of processing ICL (Zhang et al., 2005; Zhang et al., processed by the DNA helicase activity of RecQ together with 2003) and during pre-synapsis in later steps of ICL repair. the 5Ј to 3Ј exonuclease function of RecJ prior to replication To evaluate further the potential role of WRN in ICL restart (Courcelle et al., 2003). Interestingly, recent data repair/recovery processes, we examined whether WRN have shown that WRN helicase unwinds the chicken-foot interacts with other members of the RAD52 epistasis group intermediate and stimulates FEN-1 to cleave the unwound following replication arrest. We report here that WRN re- product in a structure-dependent manner (Sharma et al., 2004). localizes to a complex (or complexes) composed of RAD54, However, the precise steps and proteins involved in this process RAD51, RAD54B and ATR in response to replication arrest are still unclear (reviewed in Helleday, 2003; Hickson, 2003; induced by treatment of cells with MMC. In vitro biochemical West, 2003) and under intense investigation. analyzes reveal that WRN associates directly with RAD51 and RecQ helicases and RAD HR proteins have also been RAD54B. Based on our data we suggest a role for WRN in the documented to collaborate in DNA maintenance pathways resolution of stalled replication forks arrested by ICL, both as distinct from stalled replication fork recovery. The the candidate helicase during the early steps of processing ICL Saccharomyces cerevisiae RecQ helicase homolog, Sgs1p, as suggested by Zhang et al. (Zhang et al., 2005) and during functions together with RAD52 epistasis group members in a the later pre-synapsis step of homologous recombination. telomerase-independent alternative lengthening of telomeres (ALT) pathway (Le et al., 1999; Teng and Zakian, 1999). ALT Results pathways have also been detected in a number of immortalized Nuclear localization dynamics of WRN, RAD51, RAD54 human cell lines lacking telomerase (Bryan et al., 1997; Colgin and RAD54B in untreated cells and Reddel, 1999), and ALT cells contain ALT-associated Previous ﬂuorescence resonance energy transfer (FRET) data PML bodies consisting of promyelocytic leukaemia protein demonstrated that prolonged exposure (16-24 hours) of HeLa (PML), telomeric DNA, the telomere binding proteins TRF1 cells to MMC leads to the re-localization of RAD52 and WRN and TRF2, the RAD homologous recombination (RAD-HR) to sites of arrested replication where they physically interact

Journal of Cell Science proteins RAD50, RAD51, RAD52, MRE11, NBS1, ATR, and (Baynton et al., 2003). To study intracellular localization and the BLM and WRN helicases (Barr et al., 2003; Henson et al., re-localization dynamics as well as to identify potential protein- 2002). WRN mutations have been associated with increased protein interactions between WRN and other HR components telomere dysfunction in cells cultured from later generation upon replication fork blockage using fluorescence microscopy mice lacking the telomerase RNA component, Terc (Chang et and FRET analysis, we cloned human RAD51, RAD54 and al., 2004). These mice elicit classic WS-like premature aging RAD54B proteins as C-terminal fusions to ECFP and EYFP. manifested by precocious death, graying of the hair, Fig. 1 shows the nuclear localization of ECFP- and EYFP- osteoporosis, type II diabetes and cataracts. These data support tagged RAD51, RAD54 and RAD54B in live, untreated, a role for WRN, together with some of the RAD HR proteins, cycling HeLa cells 16-24 hours after co-transfection. ECFP- in a recombination-dependent ALT pathway. RAD51 exhibited both punctuated and rod-shaped ICL repair is poorly understood in mammalian cells, but is fluorescence patterns (Fig. 1, pictures 1 and 4), while WRN thought to involve a combination of nucleotide excision repair localized mainly in nucleoli as previously reported for (NER), translesion synthesis (TLS) and/or recombination (De endogenous WRN and both transient and stable expressing Silva et al., 2000; Zheng et al., 2003). ICLs are probably not GFP-tagged WRN (Fig. 1, picture 2) (Baynton et al., 2003; repaired until they are encountered by a DNA replication fork Opresko et al., 2004; von Kobbe and Bohr, 2002). We tried to (Akkari et al., 2000). Cells cultured from patients afflicted with generate stable cell lines that constitutively expressed low WS, BS and Nijmegen breakage syndrome (deficient for the (physiological) levels of ECFP-RAD51; however, after five NBS1 protein) all exhibit elevated sensitivity to ICLs (Pichierri weeks in selective medium, only 0.5% of the cells resistant to and Rosselli, 2004; Rosselli et al., 2003). Indeed, WRN was the selective antibiotic (geneticin) expressed ECFP-RAD51 recently shown to relocate to sites of arrested replication (Fig. 1, picture 1, insert). However, the localization pattern of induced by ICLs, where it physically and functionally interacts ECFP-RAD51 in the cells stably expressing ECFP-RAD51 with RAD52. Furthermore, WRN was found to stimulate was similar as in transiently transfected cells, and we therefore RAD52-mediated strand annealing and RAD52 enhance WRN chose to use transiently transfected cells expressing medium to helicase activity on specific substrates (Baynton et al., 2003). low levels of ECFP-RAD51 in our studies. As RAD51 has been Interestingly, RAD52 was shown to interact physically and reported to form S-phase specific nuclear foci (Tarsounas et al., functionally with the XPF/ERCC1 DNA structure-specific 2003; Tashiro et al., 1996), we co-transfected ECFP-RAD51 endonuclease involved in ICL repair (Motycka et al., 2004), with EGFP-PCNA to determine if these foci were replication WRN and HR proteins in complex after ICLs 5139

Fig. 1. Subcellular localization of WRN-, RAD51-, RAD54-, RAD54B- and PCNA-ECFP and EYFP constructs in live cycling HeLa cells. Cells co-transfected with: pictures 1-3: ECFP-RAD51 and EYFP-WRN. Inset picture 1: a stable ECFP-RAD51 expressing cell after 5 weeks in cell culture; pictures 4-6: ECFP-RAD51 and EYFP-PCNA; pictures 7-9: ECFP-RAD54B and EYFP-WRN; pictures 10-12: ECFP-PCNA and EYFP-RAD54B; pictures 13-15: ECFP-RAD54 and EYFP-WRN; pictures 16-18: ECFP- PCNA and EYFP-RAD54; pictures 19-21: ECFP-RAD51 and EYFP-RAD54; pictures 22-24: ECFP-RAD51 and EYFP-RAD54B.

forks. Tagged PCNA forms distinct foci in S phase cells that co-localize completely with endogenous PCNA at sites of bromo-deoxyuridine (BrdU) incorporation thereby representing sites of DNA synthesis (Leonhardt et al., 2000). Fig. 1 (pictures 4-6) shows that some of the RAD51 foci co-localize with PCNA in nuclear foci in unsynchronized cultures, but that there were additional RAD51 foci not co-localizing with PCNA. Cells over-expressing RAD51 have been reported to exhibit elevated levels of RAD51 foci, faster repair rates of etoposide-induced DSBs, higher levels of spontaneous HR and apoptosis, and reduced long-track HR in the hprt gene (Lundin et al., 2003). However, importantly for the present work, increased foci formation due to over-expression of the ECFP- RAD51 fusion protein did not affect the intracellular localization of either co-transfected WRN or PCNA (Fig. 1, pictures 2 and 5, respectively), indicating that over-expression of RAD51 did not itself induce replication arrest. Cells transfected with ECFP-RAD54B alone demonstrated diffuse nucleoplasmic localization with

Journal of Cell Science some minor accumulation in the nucleoli where it co- localized with co-transfected EYFP-WRN (Fig. 1, pictures 7 and 8). RAD54B and WRN also co-localized in some nucleoplasmic spots that may represent PML bodies or replication foci arrested by endogenous damage. ECFP-RAD54B did not co-localize with PCNA in replication foci (Fig. 1, pictures 10-12). When co-transfected, RAD54 and WRN co-localized in nucleoli similar to RAD54B and WRN (Fig. 1, pictures 13-15), furthermore no co-localization was seen between RAD54 and PCNA (Fig. 1, pictures 16–18). Human RAD54 and RAD54B belong to the SNF2/SW12 family of DNA-dependent ATP-ases (Swagemakers et al., 1998; Tanaka et al., 2000) and experiments suggest that yeast Rad54 plays an active role in pre-synapsis by stabilizing the Rad51 single- strand DNA filament (Mazin et al., 2003). Human RAD54 and RAD54B bind to RAD51 in vitro (Golub et al., other in vivo concurring with previous work (Golub et al., 1997; Wesoly et al., 2006) and the proteins are associated in 1997). Similar to RAD54, RAD54B re-localized into the rod- the same complex in vivo (Tanaka et al., 2000). To examine shaped RAD51 pattern when co-transfected with RAD51 (Fig. whether the tagged RAD51, RAD54 and RAD54B proteins 1, pictures 22-24) in accordance with participation in the same were functional with regard to protein-protein interactions, complex (Tanaka et al., 2002). Rad54 is reported to increase we co-transfected EYFP-RAD54 and EYFP-RAD54B with protection of DNA in a Rad51 double-stranded (ds) DNA ECFP-RAD51. RAD54 completely re-localized into a rod- filament from restriction enzyme digestion (Mazin et al., shaped fluorescent pattern also observed in cells expressing 2003); thus, the rod-shaped pattern of ECFP-RAD51 might be RAD51 alone (Fig. 1, pictures 19–21 compared to pictures 1 dsDNA-ECFP-RAD51 filaments that recruit both RAD54 and and 4), suggesting that the two proteins bind directly to each RAD54B. 5140 Journal of Cell Science 119 (24)

Co-localization of RAD51, RAD54 and RAD54B with to replication arrest and yet is minimally cytotoxic, survival WRN at the vicinity of replication centers after MMC and proliferation of HeLa cells were tested over four days induced lesions following 16 hours of exposure to 0.2 and 0.5 ␮g ml–1 MMC. To determine the appropriate mitomycin (MMC) As shown in Fig. 2A, the high percentage of live cells on day concentration that introduces DNA damage at levels leading 1 combined with an increase in the culture optical density Journal of Cell Science

Fig. 2. See next page for legend. WRN and HR proteins in complex after ICLs 5141

from days 1 to 3 demonstrated low cytotoxicity of both supporting that WRN and RAD54B re-localized to the vicinity MMC dosages (Fig. 2A). MMC concentration of 0.5 ␮g ml–1 of replication forks upon replication arrest. clearly introduced important levels of replication blocking lesions as manifested by the 3 day proliferative arrest Fluorescence resonance energy transfer (FRET) observed in the treated cells followed by the recovery to Due to the resolution limits of fluorescence microscopy, growth rates that were comparable to untreated cell cultures proteins that co-localize may not in fact interact or be in the at day 3 (Fig. 2A, lower graph), thus this dose was chosen for same complex (directly or indirectly). Therefore, we used further studies. fluorescence resonance energy transfer (FRET) to determine Following treatment with 0.5 ␮g ml–1 MMC for 16-24 hours, whether the fluorescently tagged RAD proteins and WRN were we looked at localization patterns of the RAD proteins relative in the same complex [i.e. less than 100 Å (10 nm) apart] after to WRN in transiently transfected HeLa cell cultures. Fig. 2B cellular exposure to MMC. Positive FRET is detected if the shows that the introduction of DNA intrastrand crosslinks with intensity of emitted light from the acceptor fluorochrome MMC treatment caused RAD51 (upper row), RAD54B (mid (EYFP) after excitation of the donor fluorochrome (ECFP) is row) and RAD54 (lower row) to co-localize with WRN to greater than the light emitted from cells transfected with ECFP discrete nucleoplasmic foci. Previously, WRN and RAD52 or EYFP tagged proteins alone (i.e. background levels). FRET were shown to co-localize and interact at spots resembling values within punctuated foci obtained from experiments arrested replication forks in identically treated cells (Baynton identical to those described and shown in Fig. 2B, normalized et al., 2003). At the top of upper row Fig. 2B, one cell against protein expression levels (FRETN), are presented as a expressing higher levels of both RAD51 and WRN than the dot plot in Fig. 2D. Variations in FRETN in different regions other cells in the image, but with no co-localization between of interest (ROI) are expected since they measure the dynamic the two proteins is shown. This cell, which likely represents a events in many replication sites/forks within the vicinity of cell that is not arrested in S-phase, reflects that not all cells replication centers simultaneously. Our results indicate that have achieved identical load of DNA damages after treatment WRN interacts directly or is very closely associated in either of MMC for 24 hours and furthermore that RAD51 over- a single multiprotein complex or in independent subcomplexes expression with rod-shaped fluorescence by itself does not lead with RAD51, RAD54 and RAD54B. The FRETN values to co-localization with WRN. obtained for WRN-RAD51, WRN-RAD54 and WRN- To verify that the co-localizing spots containing WRN and RAD54B are higher on average than those determined for two the RAD-proteins are in the vicinity of ‘arrested’ replicating previously published WRN interactions: WRN-RAD52 (just centers, we co-transfected HeLa cells with ECFP-RAD54 or slightly higher); and WRN-PCNA (at least twofold greater) ECFP-RAD54B, with EYFP-WRN and HcRed-PCNA. Fig. (Baynton et al., 2003). ECFP- and EYFP-tagged UNG2 2C, shows that RAD54B (pictures 1-8), RAD54 (pictures 9- proteins were used as a negative FRET control since UNG2 is 16), WRN and PCNA do co-localize in foci after MMC a monomer that localizes to replication foci (Otterlei et al., treatment in co-transfected HeLa cells, but not in untreated 1999). UNG2-ECFP and UNG2-EYFP exhibited essentially cells. In a stable EYFP-WRN expressing ALT (alternative 100% co-localization while the FRETN values using these

Journal of Cell Science lengthening of telomeres) cell line (U2OS) expressing 5-6-fold instrument settings were less than zero in more than 95% of more EYFP-WRN than endogenous WRN (data not shown), the UNG2 expressing cells. In the remaining <5% cells, FRETN we previously observed that WRN co-localized with PCNA in values were greater than zero but still fourfold lower than 28-58% of the replication foci (Opresko et al., 2004). The level values determined for WRN and RAD51, WRN and RAD54 of EYFP-WRN in this cell line is similar to the transient and WRN and RAD54B (Fig. 2D). transfected HeLa cells used in this study as determined by FRET was also used to examine functionality of the tagged ﬂuorescence intensities. Fig. 2C, pictures 17-20, show that fusion proteins with regard to protein-protein interactions. similar to in the treated HeLa cells nearly 100% of the FRET was examined after co-transfection of ECFP-RAD51 RAD54B spots co-localized with WRN and PCNA after MMC and EYFP-RAD54, and ECFP-RAD51 and EYFP-RAD54B, treatment in the stable EYFP-WRN expressing cells, which lead to re-localization of RAD54 and RAD54B into a

Fig. 2. (A) Survival and growth of HeLa cells treated with MMC. Data from untreated cells and cells exposed to MMC (0.5 and 0.2 ␮g ml–1) for 16 hours are shown. Each point represents six parallel wells. Error bars are undetectable as they are smaller than the symbols. (B) Co- localization of WRN with RAD51, RAD54B and RAD54 after treatment with MMC. Co-localization of ECFP-RAD51 and EYFP-WRN (upper row), ECFP-RAD54B and EYFP-WRN (middle row), ECFP-RAD54 and EYFP-WRN (lower row) in HeLa cells treated with MMC (0.5 ␮g ml–1) over night. (C) Re-localization of WRN, RAD54 and RAD54B to PCNA foci after MMC treatment. HeLa (pictures 1-16) and U2OS cells (pictures 17-20) were treated with MMC (0.5 ␮g ml–1) over night to arrest the cells in S-phase. HeLa cells co-transfected with ECFP-RAD54B, EYFP-WRN, HcRed-PCNA: untreated (pictures 1-4) and treated with MMC (pictures 5-8). HeLa cells co-transfected with ECFP-RAD54, EYFP-WRN, HcRed-PCNA: untreated (pictures 9-12) and treated with MMC (pictures 13-16). Stable EYFP-WRN expressing U2OS cells co- transfected with ECFP-RAD54B and HcRed-PCNA and treated with MMC (pictures 17-20). (D) FRETN values between WRN and the RAD52 epistasis proteins after MMC treatment over night, and between RAD51-RAD54 and RAD51-RAD54B. aRepresents previously published data (Baynton et al., 2003) that was taken using the same conditions and settings as the current experiments. It is presented here for comparative U 1/2 purposes. untreated cells. FRETN [FRETN=FRET/(I1 ϫ I3) ] represents FRET normalized against protein expression levels measured from intensities (I) (given as arbitrary units below 250). FRET is calculated from the mean of intensities within a region of interest containing more than 25 pixels. Within the region of interest, all pixels had intensities below 250, and the I levels were between 85-190 for the donor (ECFP) and between 55-155 (EYFP) for the acceptor, respectively. Corresponding pictures from which the data were derived are shown in Fig. 2B and Fig. 1, pictures 19-24 (RAD54-RAD51U and RAD54B–RAD51U). The background level outside foci, i.e. the co-localizing area, is 0, which is also the FRET value determined for more than 95% of foci with co-localizing UNG2-ECFP and UNG2-EYFP proteins. 5142 Journal of Cell Science 119 (24)

with DNAse. Using this approach we could IP WRN with RAD51 using anti-RAD51 antibody in cell extracts prepared from MMC treated cells, but not, or far less, from untreated cells (Fig. 3A, lanes 4 and 5). The reciprocal experiment using anti- WRN antibodies could co-IP RAD51 in extracts from both treated and untreated cells (Fig. 3A, lanes 7 and 8). We did not have sufficiently specific and/or sensitive RAD54 and RAD54B antibodies to examine co-IP WRN of with the endogenous RAD54 and RAD54B proteins. The ATR kinase is primarily responsible for eliciting the S-phase response to replication blocks. Furthermore, WRN is phosphorylated in an ATM/ATR-dependent manner and co-localizes with ATR in nuclear foci after ICL induction (Pichierri and Rosselli, 2004; Pichierri et al., 2003). Therefore, we wanted to examine whether WRN complexed with ATR in MMC treated cells by an immunoprecipitation approach. We were able to pull down, using two different ATR antibodies, what appears to be increasing amounts WRN from cell lysates prepared from MMC treated cells (Fig. 3B, lanes 3 and 4, and data not Fig. 3. Co-immunoprecipitation of WRN, RAD51 and ATR. (A) WRN is pulled shown). We also detected RAD51 with down with RAD51 (H-92) from whole cell extracts prepared from HeLa cells immunoprecipitated ATR (Fig. 3B, lanes 3 and 4). treated with MMC (0.5 ␮g ml–1), lane 5. In a WRN pulled down with the rabbit The reciprocal co-IP of ATR and WRN could not polyclonal anti-WRN (ab17987) we co-immunoprecipitated RAD51, lanes 7 and 8. easily be evaluated due to low specificity of both Mouse anti-WRN (BD) or polyclonal anti-WRN (ab17987) and anti-RAD51 of the antibodies (Fig. 3B, lanes 7 and 8). (ab1837) were used for the WBs. (B) WRN is pulled down with ATR from whole Thus, although the results for RAD54 and cell extracts prepared from HeLa cells. Rabbit polyclonal anti-ATR (ab91) and RAD54B co-IPs were inconclusive, we anti-WRN (ab200) were used for IP, and monoclonal mouse anti-WRN (BD) and nevertheless found that WRN interacts with anti-ATR (ab91) were use for WB. Input represents whole cell lysate. IgG is normal rabbit IgG (SC). RAD51 and ATR, either directly or indirectly through another as yet unidentified protein.

Journal of Cell Science Furthermore, this WRN-RAD51-ATR complex rod-shaped fluorescence pattern (Fig. 1, pictures 19-24). The forms in response to replication forks blocked by DNA ICLs. calculated FRETN values further indicate that RAD51 binds directly to both RAD54 and RAD54B proteins in vivo (Fig. WRN interacts with both RAD51 and RAD54B proteins 2D). Furthermore, these FRETN values were higher than those in vitro determined for WRN-RAD51, WRN-RAD52, WRN-RAD54 Enzyme-linked immunosorbent assays (ELISAs) using and WRN-RAD54B, which is expected since co-expression purified recombinant proteins were undertaken to further with RAD51 completely relocates RAD54 and RAD54B. characterize the interactions detected between WRN and the Thus, a large fraction of the tagged molecules present within RAD HR proteins. RAD52 was used as a positive control as it a given ROI on the filaments are likely to interact, while fewer has been shown previously to interact with both WRN of the tagged WRN and RAD proteins present in a particular (Baynton et al., 2003) and RAD51 (Shen et al., 1996). Fig. 4A ROI around foci may participate in the WRN-RAD demonstrates that WRN and RAD51 interacted regardless of interactions. This implies that the interactions between WRN which protein was used to coat the wells. The association, and the RAD51, 54 and 54B proteins at arrested replication determined by the absorbance at 490 nm, between RAD51 and sites are more dynamic than the RAD51-RAD54B and WRN was comparable to that observed between RAD51 and RAD51-RAD54 interactions. RAD52, but lower than that detected between WRN and RAD52 (Fig. 4A). Formation of a multiprotein complex containing WRN, Results from ELISAs examining RAD54 and RAD54B RAD51 and ATR in response to MMC treatment interactions with WRN (coat) are shown in Fig. 4B. RAD54B To confirm that WRN, RAD51, RAD54 and RAD54B are in the bound detectably to WRN while an association between same complex formed at blocked replication forks, we RAD54 and WRN was barely observable over background. performed co-immunoprecipitation (co-IP) experiments. Since The binding of RAD54B to WRN was less pronounced than the proteins assemble together at arrested replication sites, these RAD52, but stronger than RAD51. complexes are expected to associate with chromatin. To access Dot blot assays, in which the indicated proteins immobilized most protein complexes within the cells, also chromatin bound to PVDF membranes were incubated in a solution either with complexes, we performed immunoprecipitation experiments of or without (negative control) WRN and subsequently probed endogenous proteins in sonicated whole cell extracts digested with an anti-WRN antibody, were performed to corroborate WRN and HR proteins in complex after ICLs 5143

Fig. 4. WRN binds directly to RAD51 and RAD54B. (A) Reciprocal ELISAs showing direct binding between RAD51 and WRN. WRN, RAD52 (positive control), BSA (negative) and RAD51 were coated on microtiter plates. Binding of WRN, RAD52 and RAD51 to each other were measured as described in Materials and Methods. Values (OD490nm) for 1:1 molar ratio between the proteins are given. (B) Relative binding of RAD54B, RAD54, RAD51 and RAD52 to WRN coat in ELISA. Both results (A and B) are representative of one out of at least three independent experiments. Absorbance (OD490nm) is given as mean of duplicates adjusted for background binding to BSA in parallel duplicate wells. (C) Dot blot assay demonstrating a direct interaction between WRN and RAD51, and WRN and RAD54B. PVDF membranes with immobilized proteins were incubated in milk-solutions with or without WRN, and analyzed for WRN binding. (D) RAD51 and RAD54B do not modulate WRN helicase activity on a 12 bp bubble substrate. Native gels showing WRN (6.0 nM and 4.0 nM) mediated unwinding of a 12 bp 5Ј-labeled bubble substrate after adding increasing amounts of RAD51 (6, 12, 24 and 48 nM) and RAD54B (2, 4, 20 and 40 nM).

ELISA results. A strong interaction was detected between helicase activity on a 12 bp bubble substrate and inhibited WRN WRN and RAD52, positive binding was observed to RAD54B exonuclease activity (Baynton et al., 2003). Using substrates and a weak but positive association was detected between representing replication fork/recombination intermediates WRN and RAD51 (Fig. 4C). However, a WRN-RAD54 (bubble or fork substrates), no signiﬁcant modulation of either interaction could not be evaluated due to non-speciﬁc binding helicase or exonuclease activity was observed in the presence of the WRN antibodies to RAD54, similar to that seen with the of RAD51 or RAD54B (Fig. 4D and data not shown). negative control, BSA. Furthermore, no modulation of helicase activity was observed Ј

Journal of Cell Science The in vitro binding data demonstrate that WRN binds on a 3 -labeled (ddATP) bubble substrate, in which the directly to both RAD51 and RAD54B, in agreement with exonuclease function is physically inhibited, upon the addition FRET observations. However, even though the FRET results of RAD51 or RAD54B to assay mixtures (data not shown). indicate that WRN and RAD54 are as close as RAD54B and Thus, under these assay conditions, RAD51 and RAD54B do RAD51, and therefore, directly interact, we detected a weaker not affect WRN helicase or exonuclease activities. association between purified RAD54 and WRN proteins in ELISA. The discrepancy between the in vivo and in vitro data Discussion may be attributable to the lack of specific post-translational We report here that WRN forms either a large single modifications or a mediator protein in the in vitro system that multiprotein complex or several dynamic subcomplexes is necessary for the interaction to occur. involving RAD51, ATR, RAD54 and RAD54B at replication We find that co-transfection and over-expression of WRN forks blocked by ICLs. WRN interacts directly with RAD51 and RAD51 does not lead to co-localization of the two proteins and RAD54B; however, neither protein appears to modulate unless the cells are treated with DNA damaging agent such as WRN’s helicase or exonuclease activities on defined substrates MMC on the contrary to what is seen for RAD54-RAD51 and in vitro. These results do not preclude the possibility that RAD54B-RAD51 (Fig. 1, pictures 19-24). At the same time modulation could occur in vivo under conditions of appropriate WRN and RAD51 seem to interact in vitro in ELISA and Dot substrate, post-translational modifications or, in the presence blot assay. An explanation for this may be that WRN need of an essential mediator protein. Together with previous studies some sort of post-translational modifications, such as showing that WRN interacts with RAD52 (Baynton et al., phosphorylation, to interact with RAD51 and that WRN 2003), NBS1 in the MRN complex (Cheng et al., 2004) and isolated from insect cell cultures are sufficiently Fen-1 (Brosh et al., 2001; Sharma et al., 2004), our results phosphorylated to mediate such interaction also in vitro. provide additional support of a role for WRN in recombination associated with replication restart. RAD51 and RAD54B do not modulate WRN activity on Similar to WRN, BLM helicase also associates with RAD51 fork or bubble substrates and ATR, and is phosphorylated by ATR (Davies et al., 2004; Several of the reported physical interactions between WRN and Wu et al., 2001). BS cells, like WS cell cultures, exhibit its various protein partners modify WRN activity on defined sensitivity to the DNA crosslinking agent mitomycin C (MMC) substrates. Previously, we found that RAD52 stimulated WRN (Hook et al., 1984), suggesting that BLM also functions in ICL 5144 Journal of Cell Science 119 (24)

repair. WRN and BLM co-localize in untreated cells and annealing (SSA). XPF/ERCC1 also interacts physically and interact physically and functionally in vitro (von Kobbe et al., functionally with RAD52, resulting in the stimulation of the 2002). Thus, these two RecQ helicases may, in some instances, XPF/ERCC1 DNA structure-specific endonuclease function co-operate or provide back up functions in certain repair and inhibition of RAD52 strand annealing activity (Motycka responses, including possibly ATR-mediated ICL repair. et al., 2004). We previously demonstrated that RAD52 However, both helicases clearly execute non-overlapping, stimulates WRN helicase mediated unwinding of a bubble unique functions. Both BS and WS cell cultures exhibit substrate and WRN enhances RAD52 strand annealing increased crossover-associated recombination events and properties (Baynton et al., 2003). Thus, WRN may affect ICL reduced recombination efficiency, but only BS cells repair at several levels by performing helix opening in co- specifically demonstrate elevated sister chromatid exchanges operation with RPA and Pso4complex in accordance with the (SCEs) (Hickson, 2003). model suggested by Zhang et al. (Zhang et al., 2005; Zhang et Several lines of in vivo experimental evidence link WRN to al., 2003), prior to uncoupling of the ICL by XPF/ERCC1 stalled replication forks. For example, WRN relocates from endonuclease and strand annealing/invasion by the RAD HR nucleoli to nucleoplasmic foci after treatment with DNA proteins. Our data showing interactions of WRN with damaging agents where it co-localizes with NBS1, RAD51 and complexes containing ATR, RAD51, 52, 54 and 54B support RPA, respectively (Cheng et al., 2004; Sakamoto et al., 2001). a role for WRN also in the later HR steps of ICL-repair. Both WRN and NBS1 is phosphorylated in an ATR/ATM- dependent manner following replication arrest (Pichierri and Materials and Methods Franchitto, 2004; Pichierri et al., 2003). Furthermore, NBS1 of Purification of recombinant proteins the MRN assembly, in collaboration with FANCD2, RAD52 and WRN were purified as described (Baynton et al., 2003; Opresko et al., 2002). RAD51 was a generous gift from Ian Hickson. Flag-RAD54 and RAD54B participates in one of two parallel ATR controlled ICL induced were prepared as described in Sigurdsson et al. (Sigurdsson et al., 2002) and Sehorn pathways, the second pathway involving CHK1 (Pichierri and et al. (Sehorn et al., 2004), respectively. Rosselli, 2004). Co-operation between these two pathways leads to S phase checkpoint specific activation. Recent data Cloning of fusion proteins Cloning of fluorescent-tagged expression constructs pEYFP-WRN, pECFP- strongly indicate that WRN and the MRN complex do act in a RAD52, pECFP-PCNA, pUNG2-ECFP and pUNG2-EYFP has been previously common pathway upon replication arrest (Franchitto and described (Baynton et al., 2003; Aas et al., 2003). RAD51 was PCR amplified from Pichierri, 2004). A model has been proposed describing how pFB530 (Benson et al., 1994) and ligated into the PstI/BamHI restriction sites of pECFP-C1 vector (Clontech). RAD54 was PCR amplified from pBluescript WRN and the MRN complex could co-operate in resolving (Sigurdsson et al., 2002) and cloned into the KpnI site of pECFP-C1 and pEYFP- abnormal structures at stalled replication forks during S-phase. C1 vectors. RAD54B in pUC18 (Sehorn et al., 2004) was PCR amplified and cloned Indeed, the interaction between NBS1 and WRN, combined into the XhoI/XmaI site of pECFP-C1 and pEYFP-C1 vectors. All constructs were verified by sequencing. HcRed-PCNA and stable expressing EYFP-WRN U2OS with results from the current study demonstrating that WRN cells have been previously described (Opresko et al., 2004). and ATR are present in the same complex in response to ICL induction, support a role for WRN in the NBS branch of the Cell culture ATR controlled ICL repair response, possibly together with Cells were transfected with the different constructs of fusion proteins using the calcium phosphate- method (Profection Promega) according to the manufacturer’s Journal of Cell Science RAD51, RAD54, RAD54B and RAD52. RAD51 null mutation recommendations. Stable ECFP-RAD51 expressing cells (HeLa) were cultured in is embryonic lethal in mice while the cellular phenotypes of DMEM media (Gibco) containing 10% fetal calf serum, gentamycin (100 ␮g ml–1, null RAD52 and RAD54 mutants in vertebrates are less severe, Gibco), glutamine, fungizone and geneticin [G418, 400 ␮g ml–1 (Invitrogen), added however they have been reported to have reduced HR 2 days after transfection]. For transient transfections the cells were examined after 16-24 hours, and no geneticin was added. Untransfected cells were cultured in the frequency (both) and increased sensitivity to IR, the inter- same media without geneticin. strand cross-linking agent mitomycin C (MMC) and methyl methanesulfonate (MMS) (only RAD54–/–) (Essers et al., Cell survival and growth in the presence of MMC HeLa cells were seeded into four 96 well plates at three different cell densities. A 1997; Rijkers et al., 1998). dose response curve to MMC was determined for each cell concentration. All plates Recent attempts to reconstitute psoralen ICL repair in vitro were incubated for 16 hours before being washed three times with PBS. At that time have revealed that PCNA is a stimulatory factor while RPA is (day 1), the first plate was harvested using the MTT assay (Mosmann, 1983). Fresh essential at the incision step. Furthermore, RPA stimulates medium was added to the remaining plates, which were further incubated for 1 to 3 days before being assayed for cell proliferation and survival by the MTT assay. helicases, which in turn, create an open bubble region proximal The cell density permitting growth to be followed for four days without saturation to the ICL prior to incision (Zhang et al., 2003; Zhang et al., of the OD570 signal after MTT addition in the control wells (no MMC added) were 2002). Candidate human helicases include WRN, BLM and selected. The mean OD570 from 6 wells was used to calculate cells survival and plot ␮ –1 RECQ1, all which bind to and are stimulated by RPA (Brosh the growth after addition of 0.5 g ml MMC. et al., 2000; Brosh et al., 1999; Cui et al., 2004; Cui et al., Confocal microscopy and FRET measurements 2003). In fact, recently WRN was found to interact with the Fluorescent images of living cells co-transfected with ECFP, EYFP and HcRed constructs (1 ␮m thickness) were produced using a Zeiss LSM 510 laser scanning pre-mRNA splicing Pso4-complex, and both WRN and the ϫ Pso4-complex were found to be required for ICL repair in an microscope equipped with a Plan-Apochromate 63 , 1.4 oil immersion objective. FRET, if I2 – I1[ID2/ID1] – I3[IA2/IA3]>0, where I represents intensities in three in vitro assay (Zhang et al., 2005). They propose a model where channels given in arbitrary units, was determined as described (Baynton et al., WRN is the candidate helicase involved in the early steps of 2003). The FRET values were normalized to account for differences in the respective fluorochrome expression levels using the following equation: normalized processing ICL. Additional requirements for psoralen ICL 1/2 FRET (FRETN) = FRET/(I1 ϫ I3) according to Xia and Liu (Xia and Liu, 2001). repair include MutS␤, together with the ERCC1/XPF Three color images were taken with three consecutive scans using the following endonuclease, during initial lesion processing (Zhang et al., settings: ECFP-RAD54B excitation at ␭=458 nm, detection at ␭=470-500 nm, 2002). The endonuclease activity is essential for removing long EYFP-WRN excitation at ␭=488 nm, at ␭=500-550 and HcRed-PCNA excitation at Ј ␭=543 nm, detection at ␭>585 on a Zeiss LSM Meta laser scanning microscope. non-homologous stretches from DNA 3 -OH ends during All co-transfection experiments were repeated at least three times and included an targeted homologous recombination and single strand analysis of more than 50 cells. WRN and HR proteins in complex after ICLs 5145

Antibodies Software (Molecular Dynamics). A 12 bp bubble substrate with 19 bp flanking arms Anti-WRN mouse monoclonal antibody (Ab) (BD Biosciences Pharmigen, USA), (Baynton et al., 2003) and a long fork substrate with a 15 bp fork and 34 bp duplex anti WRN polyclonal Ab (ab200 and ab17987), anti-GFP polyclonal (ab290), anti- arm (X/Y substrate) were used as substrates (Opresko et al., 2002). ATR polyclonal (ab91) and anti-RAD51 monoclonal (ab1839) (Abcam, UK) were used in these studies. Anti-RAD51 (SC H-92), anti-RAD52 (SC H-300), anti- This work was done partly during the different contributors’ stay at RAD54 (SC H-152), RAD54B (SC N-16), RAD54B (SC K-17), rabbit and goat the Laboratory of Molecular Gerontology, National Institute on IgG, and all polyclonal antibodies were obtained from Santa Cruz Biotechnology, Inc. (USA). In addition, anti RAD54 and anti RAD54B rabbit antiserum made Aging, NIH, USA and partly at Department of Cancer Research and against purified Flag-RAD54 and RAD54B proteins were used in the ELISA assay. Molecular Medicine, NTNU. We would like to thank Mette Sørensen, IgG cross-linking to protein A magnetic beads was done according to New England Bruno Monterotti and Knut Lauritzen, NTNU, for technical Biolabs (USA) assistance, Ian Hickson, Cancer Research UK Laboratories, Oxford, UK for the generous gift of purified RAD51, and Patrick Sung, Yale Cell extracts, immune-precipitation (IP) and western blot University School of Medicine, New Haven, CT, USA for RAD54- analysis (WB) and RAD54B-expressing plasmids. We want to thank Patricia Cell pellets were snap frozen in liquid nitrogen before preparing the whole cell lysates. All successive steps were done with ice-cold buffers. Cell pellets were re- Opresko and Wen-Hsing Cheng for critical reading of the manuscript. suspended in 1ϫ the packed cell volume (PCV) with buffer I (10 mM Tris-HCl, This work is supported by The Research Council of Norway, The pH 7.8, 100 mM KCl) to which an equal volume of buffer II (10 mM Tris-HCl, Cancer Fund at St Olav’s Hospital, Trondheim, Norway, 2005 pH 8.0, 100 mM KCl, 40% (v/v) glycerol, 0.5% Nonidet P-40, 10 mM EGTA, 10 Research Fund of University of Ulsan, Korea and Intramural Research ® mM MgCl2, 2 mM DTT, Complete protease inhibitor, 1 mM PMSF) was added. Program of the NIH, National Institute on Aging, USA. 1 ␮l (200 U ␮l–1) of OmnicleaveTM Endonuclease (Epicentre Technologies, WI, USA) was added per 800 ␮l cell suspension. The cells were briefly sonicated until complete destruction was achieved as determined by microscopy. Total protein References concentrations were measured using the Bio-Rad protein assay. Cell extracts were Aas, P. A., Otterlei, M., Falnes, P. O., Vagbo, C. B., Skorpen, F., Akbari, M., snap frozen in liquid nitrogen and stored in aliquots at –80°C. For each IP, Sundheim, O., Bjoras, M., Slupphaug, G., Seeberg, E. et al. (2003). Human and approximately 500 ␮g of total cell lysate was incubated with the different bacterial oxidative demethylases repair alkylation damage in both RNA and DNA. antibodies covalently linked to protein-A beads, at 4°C over night. The beads were Nature 421, 859-863. washed 5 times with 0.5 ml buffer mix (1:1) of buffer I: buffer II with 5 mM MgCl Akkari, Y. M., Bateman, R. L., Reifsteck, C. A., Olson, S. B. and Grompe, M. (2000). 2 DNA replication is required to elicit cellular responses to psoralen-induced DNA and 400 U Omnicleave per 50 ml. After washing, the beads were re-suspended in ␮ TM interstrand cross-links. Mol. Cell. Biol. 20, 8283-8289. 20 l NuPAGE loading buffer, heated, and loaded on 4-12% Bis-Tris-HCl Alexeev, A., Mazin, A. and Kowalczykowski, S. C. (2003). Rad54 protein possesses NuPAGETM ready gels (Invitrogen) and the separated, immuneprecipitated proteins TM chromatin-remodeling activity stimulated by the Rad51-ssDNA nucleoprotein filament. were subsequently transferred to PVDF membranes (Immobilon , Millipore). Nat. Struct. Biol. 10, 182-186. The membranes were blocked (5% dry milk) and the primary rabbit polyclonal or Barr, S. M., Leung, C. G., Chang, E. E. and Cimprich, K. A. (2003). ATR kinase mouse monoclonal antibodies were diluted in dry milk (1%) in PBST (PBS with activity regulates the intranuclear translocation of ATR and RPA following ionizing 0.1% Tween 20). Peroxidase-labeled (HRP) goat anti-rabbit IgG, HRP-horse anti- radiation. Curr. Biol. 13, 1047-1051. mouse IgG (Vector Lab, USA), HRP-swine-anti-rabbit IgG or HRP-goat-anti- Baynton, K., Otterlei, M., Bjoras, M., von Kobbe, C., Bohr, V. A. and Seeberg, E. mouse IgG (DAKO, Denmark) were used as secondary antibodies. Membranes (2003). WRN interacts physically and functionally with the recombination mediator were treated with ECL chemiluminescence reagent (ECLTM, Amersham protein RAD52. J. Biol. Chem. 278, 36476-36486. Biosciences) and the bands were visualized by exposing the membranes to Benson, F. E., Stasiak, A. and West, S. C. (1994). Purification and characterization of HyperfilmTM, ECLTM. the human Rad51 protein, an analogue of E. coli RecA. EMBO J. 13, 5764-5771. Brosh, R. M., Jr, Li, J. L., Kenny, M. K., Karow, J. K., Cooper, M. P., Kureekattil, Enzyme-linked immunosorbent assays (ELISAs) R. P., Hickson, I. D. and Bohr, V. A. (2000). Replication protein A physically interacts with the Bloom’s syndrome protein and stimulates its helicase activity. J. Biol. Chem. All assays were performed in triplicate. Microtiter plates were coated with 1.5 pmol 275, 23500-23508. RAD51, WRN or RAD52 diluted in 100 ␮l PBS at 4° over night. The wells were Journal of Cell Science ␮ Brosh, R. M., Jr, Orren, D. K., Nehlin, J. O., Ravn, P. H., Kenny, M. K., Machwe, blocked with 200 l 2% BSA in 1% Tween 20 in PBS for 2 hours at room A. and Bohr, V. A. (1999). Functional and physical interaction between WRN helicase ␮ temperature (RT). 100 l of equimolar, two- and fivefold serial dilutions [in 2% and human replication protein A. J. Biol. Chem. 274, 18341-18350. BSA, 0.1% Tween20 in PBS (PBST)] of RAD51, RAD52, RAD54, RAD54B or Brosh, R. M., Jr, von Kobbe, C., Sommers, J. A., Karmakar, P., Opresko, P. L., WRN were added to the coated wells and also to wells containing only BSA, and Piotrowski, J., Dianova, I., Dianov, G. L. and Bohr, V. A. (2001). Werner syndrome the plates were further incubated for 1 hour at RT. Control wells for antibody protein interacts with human flap endonuclease 1 and stimulates its cleavage activity. specificity and reactivity were included. The wells were then washed five times with EMBO J. 20, 5791-5801. 200 ␮l PBST and thereafter, washed three times with 200 ␮l PBST between Bryan, T. M., Englezou, A., Dalla-Pozza, L., Dunham, M. A. and Reddel, R. R. consecutive steps. Primary rabbit antibodies against WRN (ab200 1:1500), RAD51 (1997). Evidence for an alternative mechanism for maintaining telomere length in (SC H92 1:200), RAD52 (SC H300 1:200), RAD54 (rabbit antiserum 1:500), human tumors and tumor-derived cell lines. Nat. Med. 3, 1271-1274. RAD54B (rabbit antiserum 1:3000) and secondary HRP-goat anti rabbit antibody Chang, S., Multani, A. S., Cabrera, N. G., Naylor, M. L., Laud, P., Lombard, D., (vector 1:5000) were diluted in PBS and 1 mg ml–1 BSA and successively added Pathak, S., Guarente, L. and DePinho, R. A. (2004). Essential role of limiting for 1 hour at RT (100 ␮l per well). Finally, the substrate (o-phenylenediamine, telomeres in the pathogenesis of Werner syndrome. Nat. Genet. 36, 877-882. Dacopatts A/S) was added for 10-30 minutes and stopped with addition of sulfuric Cheng, W. H., Von Kobbe, C., Opresko, P. L., Arthur, L. M., Komatsu, K., Seidman, acid according to manufacturer’s recommendations. Data are presented as mean of M. M., Carney, J. P. and Bohr, V. A. (2004). Linkage between werner syndrome protein and the Mre11 complex via Nbs1. J. Biol. Chem. 279, 21169-21176. duplicates adjusted for background binding to BSA. Colgin, L. M. and Reddel, R. R. (1999). Telomere maintenance mechanisms and cellular immortalization. Curr. Opin. Genet. Dev. 9, 97-103. Dot blot Constantinou, A., Tarsounas, M., Karow, J. K., Brosh, R. M., Bohr, V. A., Hickson, 500 ng WRN, RAD52, RAD54, RAD54B and BSA were immobilized on a PVDF I. D. and West, S. C. (2000). Werner’s syndrome protein (WRN) migrates Holliday membrane (ImmobilonTM, Millipore). The filters were dried, blocked with 5% dry junctions and co-localizes with RPA upon replication arrest. EMBO Rep. 1, 80-84. milk in PBS and incubated either with 500 ng ml–1 WRN in PBST and 1% dry milk, Cooper, M. P., Machwe, A., Orren, D. K., Brosh, R. M., Ramsden, D. and Bohr, V. or with PBST and 1% dry milk alone (to distinguish between specific and unspecific A. (2000). Ku complex interacts with and stimulates the Werner protein. Genes Dev. binding of WRN and the antibodies) at 4°C overnight. After washing, the 14, 907-912. membranes were incubated with the mouse monoclonal anti-WRN (BD, 1:250) in Courcelle, J., Donaldson, J. R., Chow, K. H. and Courcelle, C. T. (2003). DNA PBST and 1% dry milk at 4°C overnight and developed as an ordinary western blot damage-induced replication fork regression and processing in Escherichia coli. Science (see above). 299, 1064-1067. Cui, S., Arosio, D., Doherty, K. M., Brosh, R. M., Jr, Falaschi, A. and Vindigni, A. WRN helicase and exonuclease reactions (2004). Analysis of the unwinding activity of the dimeric RECQ1 helicase in the ␮ presence of human replication protein A. Nucleic Acids Res. 32, 2158-2170. Reactions (20 or 30 l) were performed in standard reaction buffer as described Cui, S., Klima, R., Ochem, A., Arosio, D., Falaschi, A. and Vindigni, A. (2003). previously (Opresko et al., 2002) unless otherwise indicated. DNA substrate Characterization of the DNA-unwinding activity of human RECQ1, a helicase concentrations were 2 nM and protein concentrations were as indicated in the figure specifically stimulated by human replication protein A. J. Biol. Chem. 278, 1424-1432. legends. The reactions were initiated by WRN addition and incubated at 37°C for Davies, S. L., North, P. S., Dart, A., Lakin, N. D. and Hickson, I. D. (2004). 15 minutes. To visualize the helicase product, native stop dye was added to the Phosphorylation of the Bloom’s syndrome helicase and its role in recovery from S- reaction mixture, which was analyzed on a 12% native polyacrylamide gel. Products phase arrest. Mol. Cell. Biol. 24, 1279-1291. were visualized by Phosphorimager and were quantitated with ImageQuant De Silva, I. U., McHugh, P. J., Clingen, P. H. and Hartley, J. A. (2000). Defining the 5146 Journal of Cell Science 119 (24)

roles of nucleotide excision repair and recombination in the repair of DNA interstrand depends on ATR-CHK1 and ATR-NBS1-FANCD2 pathways. EMBO J. 23, 1178- cross-links in mammalian cells. Mol. Cell. Biol. 20, 7980-7990. 1187. Essers, J., Hendriks, R. W., Swagemakers, S. M., Troelstra, C., de Wit, J., Bootsma, Pichierri, P., Rosselli, F. and Franchitto, A. (2003). Werner’s syndrome protein is D., Hoeijmakers, J. H. and Kanaar, R. (1997). Disruption of mouse RAD54 reduces phosphorylated in an ATR/ATM-dependent manner following replication arrest ionizing radiation resistance and homologous recombination. Cell 89, 195-204. and DNA damage induced during the S phase of the cell cycle. Oncogene 22, 1491- Franchitto, A. and Pichierri, P. (2004). Werner syndrome protein and the MRE11 1500. complex are involved in a common pathway of replication fork recovery. Cell Cycle Rijkers, T., Van Den Ouweland, J., Morolli, B., Rolink, A. G., Baarends, W. M., Van 3, 1331-1339. Sloun, P. P., Lohman, P. H. and Pastink, A. (1998). Targeted inactivation of mouse Fukuchi, K., Martin, G. M. and Monnat, R. J., Jr (1989). Mutator phenotype of Werner RAD52 reduces homologous recombination but not resistance to ionizing radiation. syndrome is characterized by extensive deletions. Proc. Natl. Acad. Sci. USA 86, 5893- Mol. Cell. Biol. 18, 6423-6429. 5897. Rosselli, F., Briot, D. and Pichierri, P. (2003). The Fanconi anemia pathway and the Golub, E. I., Kovalenko, O. V., Gupta, R. C., Ward, D. C. and Radding, C. M. (1997). DNA interstrand cross-links repair. Biochimie 85, 1175-1184. Interaction of human recombination proteins Rad51 and Rad54. Nucleic Acids Res. 25, Sakamoto, S., Nishikawa, K., Heo, S. J., Goto, M., Furuichi, Y. and Shimamoto, A. 4106-4110. (2001). Werner helicase relocates into nuclear foci in response to DNA damaging Grigorova, M., Balajee, A. S. and Natarajan, A. T. (2000). Spontaneous and X-ray- agents and co-localizes with RPA and Rad51. Genes Cells 6, 421-430. induced chromosomal aberrations in Werner syndrome cells detected by FISH using Salk, D. (1982). Werner’s syndrome: a review of recent research with an analysis of chromosome-specific painting probes. Mutagenesis 15, 303-310. connective tissue metabolism, growth control of cultured cells, and chromosomal Helleday, T. (2003). Pathways for mitotic homologous recombination in mammalian aberrations. Hum. Genet. 62, 1-5. cells. Mutat. Res. 532, 103-115. Sehorn, M. G., Sigurdsson, S., Bussen, W., Unger, V. M. and Sung, P. (2004). Human Henson, J. D., Neumann, A. A., Yeager, T. R. and Reddel, R. R. (2002). Alternative meiotic recombinase Dmc1 promotes ATP-dependent homologous DNA strand lengthening of telomeres in mammalian cells. Oncogene 21, 598-610. exchange. Nature 429, 433-437. Hickson, I. D. (2003). RecQ helicases: caretakers of the genome. Nat. Rev. Cancer 3, Sharma, S., Otterlei, M., Sommers, J. A., Driscoll, H. C., Dianov, G. L., Kao, H. I., 169-178. Bambara, R. A. and Brosh, R. M., Jr (2004). WRN helicase and FEN-1 form a Hook, G. J., Kwok, E. and Heddle, J. A. (1984). Sensitivity of Bloom syndrome complex upon replication arrest and together process branch migrating DNA Structures fibroblasts to mitomycin C. Mutat. Res. 131, 223-230. associated with the replication fork. Mol. Biol. Cell 15, 734-750. Hsu, H. L., Gilley, D., Galande, S. A., Hande, M. P., Allen, B., Kim, S. H., Li, G. C., Shen, Z., Cloud, K. G., Chen, D. J. and Park, M. S. (1996). Specific interactions Campisi, J., Kohwi-Shigematsu, T. and Chen, D. J. (2000). Ku acts in a unique way between the human RAD51 and RAD52 proteins. J. Biol. Chem. 271, 148-152. at the mammalian telomere to prevent end joining. Genes Dev. 14, 2807-2812. Sigurdsson, S., Van Komen, S., Petukhova, G. and Sung, P. (2002). Homologous DNA Huang, S., Li, B., Gray, M. D., Oshima, J., Mian, I. S. and Campisi, J. (1998). The pairing by human recombination factors Rad51 and Rad54. J. Biol. Chem. 277, 42790- premature ageing syndrome protein, WRN, is a 3Јr5Ј exonuclease. Nat. Genet. 20, 42794. 114-116. Swagemakers, S. M., Essers, J., de Wit, J., Hoeijmakers, J. H. and Kanaar, R. (1998). Kamath-Loeb, A. S., Johansson, E., Burgers, P. M. and Loeb, L. A. (2000). Functional The human RAD54 recombinational DNA repair protein is a double-stranded DNA- interaction between the Werner Syndrome protein and DNA polymerase delta. Proc. dependent ATPase. J. Biol. Chem. 273, 28292-28297. Natl. Acad. Sci. USA 97, 4603-4608. Tan, T. L., Kanaar, R. and Wyman, C. (2003). Rad54, a Jack of all trades in homologous Le, S., Moore, J. K., Haber, J. E. and Greider, C. W. (1999). RAD50 and RAD51 define recombination. DNA Repair 2, 787-794. two pathways that collaborate to maintain telomeres in the absence of telomerase. Tanaka, K., Hiramoto, T., Fukuda, T. and Miyagawa, K. (2000). A novel human rad54 Genetics 152, 143-152. homologue, Rad54B, associates with Rad51. J. Biol. Chem. 275, 26316-26321. Lebel, M., Spillare, E. A., Harris, C. C. and Leder, P. (1999). The Werner syndrome Tanaka, K., Kagawa, W., Kinebuchi, T., Kurumizaka, H. and Miyagawa, K. (2002). gene product co-purifies with the DNA replication complex and interacts with PCNA Human Rad54B is a double-stranded DNA-dependent ATPase and has biochemical and topoisomerase I. J. Biol. Chem. 274, 37795-37799. properties different from its structural homolog in yeast, Tid1/Rdh54. Nucleic Acids Leonhardt, H., Rahn, H. P., Weinzierl, P., Sporbert, A., Cremer, T., Zink, D. and Res. 30, 1346-1353. Cardoso, M. C. (2000). Dynamics of DNA replication factories in living cells. J. Cell Tarsounas, M., Davies, D. and West, S. C. (2003). BRCA2-dependent and independent Biol. 149, 271-280. formation of RAD51 nuclear foci. Oncogene 22, 1115-1123. Lieber, M. R., Ma, Y., Pannicke, U. and Schwarz, K. (2003). Mechanism and regulation Tashiro, S., Kotomura, N., Shinohara, A., Tanaka, K., Ueda, K. and Kamada, N. of human non-homologous DNA end-joining. Nat. Rev. Mol. Cell. Biol. 4, 712-720. (1996). S phase specific formation of the human Rad51 protein nuclear foci in Lundin, C., Schultz, N., Arnaudeau, C., Mohindra, A., Hansen, L. T. and Helleday, lymphocytes. Oncogene 12, 2165-2170. T. (2003). RAD51 is involved in repair of damage associated with DNA replication in Teng, S. C. and Zakian, V. A. (1999). Telomere-telomere recombination is an efficient

Journal of Cell Science mammalian cells. J. Mol. Biol. 328, 521-535. bypass pathway for telomere maintenance in Saccharomyces cerevisiae. Mol. Cell. Martin, G. M. (1978). Genetic syndromes in man with potential relevance to the Biol. 19, 8083-8093. pathobiology of aging. Birth. Defects Orig. Artic. Ser. 14, 5-39. von Kobbe, C. and Bohr, V. A. (2002). A nucleolar targeting sequence in the Werner Martin, G. M., Sprague, C. A. and Epstein, C. J. (1970). Replicative life-span of syndrome protein resides within residues 949-1092. J. Cell Sci. 115, 3901-3907. cultivated human cells. Effects of donor’s age, tissue, and genotype. Lab. Invest. 23, von Kobbe, C., Karmakar, P., Dawut, L., Opresko, P., Zeng, X., Brosh, R. M., Jr, 86-92. Hickson, I. D. and Bohr, V. A. (2002). Colocalization, physical, and functional Mazin, A. V., Alexeev, A. A. and Kowalczykowski, S. C. (2003). A novel function of interaction between Werner and Bloom syndrome proteins. J. Biol. Chem. 277, 22035- Rad54 protein. Stabilization of the Rad51 nucleoprotein filament. J. Biol. Chem. 278, 22044. 14029-14036. Wesoly, J., Agarwal, S., Sigurdsson, S., Bussen, W., Van Komen, S., Qin, J., van Steeg, Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: H., van Benthem, J., Wassenaar, E., Baarends, W. M. et al. (2006). Differential application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63. contributions of mammalian Rad54 paralogs to recombination, DNA damage repair, Motycka, T. A., Bessho, T., Post, S. M., Sung, P. and Tomkinson, A. E. (2004). Physical and meiosis. Mol. Cell. Biol. 26, 976-989. and functional interaction between the XPF/ERCC1 endonuclease and hRad52. J. Biol. West, S. C. (2003). Molecular views of recombination proteins and their control. Nat. Chem. 279, 13634-13639. Rev Mol. Cell. Biol. 4, 435-445. Opresko, P. L., Cheng, W. H., von Kobbe, C., Harrigan, J. A. and Bohr, V. A. (2003). Wu, L., Davies, S. L., Levitt, N. C. and Hickson, I. D. (2001). Potential role for the Werner syndrome and the function of the Werner protein; what they can teach us about BLM helicase in recombinational repair via a conserved interaction with RAD51. J. the molecular aging process. Carcinogenesis 24, 791-802. Biol. Chem. 276, 19375-19381. Opresko, P. L., Laine, J. P., Brosh, R. M., Jr, Seidman, M. M. and Bohr, V. A. (2001). Xia, Z. and Liu, Y. (2001). Reliable and global measurement of fluorescence resonance Coordinate action of the helicase and 3Ј to 5Ј exonuclease of Werner syndrome protein. energy transfer using fluorescence microscopes. Biophys. J. 81, 2395-2402. J. Biol. Chem. 276, 44677-44687. Yu, C. E., Oshima, J., Fu, Y. H., Wijsman, E. M., Hisama, F., Alisch, R., Matthews, Opresko, P. L., Otterlei, M., Graakjaer, J., Bruheim, P., Dawut, L., Kolvraa, S., May, S., Nakura, J., Miki, T., Ouais, S. et al. (1996). Positional cloning of the Werner’s A., Seidman, M. M. and Bohr, V. A. (2004). The Werner syndrome helicase and syndrome gene. Science 272, 258-262. exonuclease cooperate to resolve telomeric D loops in a manner regulated by TRF1 Zhang, N., Kaur, R., Lu, X., Shen, X., Li, L. and Legerski, R. J. (2005). The Pso4 and TRF2. Mol. Cell 14, 763-774. mRNA splicing and DNA repair complex interacts with WRN for processing of DNA Opresko, P. L., von Kobbe, C., Laine, J. P., Harrigan, J., Hickson, I. D. and Bohr, V. interstrand cross-links. J. Biol. Chem. 280, 40559-40567. A. (2002). Telomere-binding protein TRF2 binds to and stimulates the Werner and Zhang, N., Lu, X. and Legerski, R. J. (2003). Partial reconstitution of human interstrand Bloom syndrome helicases. J. Biol. Chem. 277, 41110-41119. cross-link repair in vitro: characterization of the roles of RPA and PCNA. Biochem. Otterlei, M., Warbrick, E., Nagelhus, T. A., Haug, T., Slupphaug, G., Akbari, M., Biophys. Res. Commun. 309, 71-78. Aas, P. A., Steinsbekk, K., Bakke, O. and Krokan, H. E. (1999). Post-replicative Zhang, N., Lu, X., Zhang, X., Peterson, C. A. and Legerski, R. J. (2002). hMutSbeta base excision repair in replication foci. EMBO J. 18, 3834-3844. is required for the recognition and uncoupling of psoralen interstrand cross-links in Pichierri, P. and Franchitto, A. (2004). Werner syndrome protein, the MRE11 complex vitro. Mol. Cell. Biol. 22, 2388-2397. and ATR: menage-a-trois in guarding genome stability during DNA replication? Zheng, H., Wang, X., Warren, A. J., Legerski, R. J., Nairn, R. S., Hamilton, J. W. BioEssays 26, 306-313. and Li, L. (2003). Nucleotide excision repair- and polymerase eta-mediated error- Pichierri, P. and Rosselli, F. (2004). The DNA crosslink-induced S-phase checkpoint prone removal of mitomycin C interstrand cross-links. Mol. Cell. Biol. 23, 754-761.